Document QkVQyQBoeqXNrN5R9R7rd8RN7

KJ /' \ hy ' Ja/vary l%4 So. 17 N THIS ISSUE: THE DETERMINATION OF TRACE METALS LN BLOOD AND URINE BY ATOMIC ABSORPTION' SPECTROPHOTOMETRY.............................................. 1 THE DETERMINATION OF CERTAIN TOXICOLOGICAL TRACE METALS BY ATOMIC ABSORPTION SPECTROPHOTOMETRY...............................................7 TTIE DETERMINATION OF TRACE METALS IN BLOOO AND URINE BY ATOMIC ABSORPTION SPECTROPHOTOMETRY* falter SJatia and Sabina Sprague PrrtinElmrr Carpe ratio* Narualk. Corottcticut The determination at tbe low major metallic constituent* of biological syatemaodium, potassium, calcium and magnesium- by atomic absorption spectrophotometry it well established and in routine use in many Islmratories Hut the atomic absorption method a)o it ideally suited lo I be determination of trace metals More than a year ago Nillis published a de termination of zinc and cadiuta in urine (1) requiring basic equipment and a very direct procedure. Lair* an organic extraction procedurr. be determined lead, bismuth. mercury tad nickel in urine samples. Another worker. Feldman, also oed an ettraction technique for determining mercury in urine (2). In adiitioo, chromium, cabrium, cobalt, lead andthallium at toxicological concentra tions litre all been determined in blood and urine (3). The procedure is short and direct for metal* that occur at relatively high levels it serum and urine For example, urine samples with metals sorb at copper. iron tod line ia the nnge from 0 5 to 2 *g per ml are determined directly with no sample preparation at all. Serum sample* must be deprotetnired since the protein precipitates in the burner tnd causes clogging. All three metals can lie determined without interferences against standards containing only ibe desired metal. Excluding protein prec Ipitstion, where required, each metal determination la ta ansi y si a can be done la less tbaa five minutes. SERUM IRON SI I serum samples were analyzed for iron Sample* of shout 2 ml of 14nod W*>fa diluted 1:1 with 20% Iricbloroar etir ar id and ( enlufuged They % ere then burned Ia a PerltlaF.lmer Model 303 andlbe ahsorptioa re<orlcd at ibe inn resonance line The iron concentration la the samples was determined from a working curve fxejmnd from the absorption of iron Stand ards m a dilute atid solution The results sre compared in Tibia I with colon metric analyses perfumed by the llamsay technique (q) at the Nailer lleed Army Institute of Hesearrh which supplied the staples. ! _51!,'. enasri Part of this mstensl was (resented at the ('linn al Chemical Congress, Detroit, Aug. 1963. .juftzm,neauaiaro.fri^.aws^j-jwaij***jaj.. . ajw'jw: ..u. -a.up* o ... ""m 'ti* .'-vroB&raam1 ft -1 . I^Tra^ji.1 mTl~^l?0 J5*^^^^jp^^y^<rej|j)i|i.lM^C^irA<liqfg^liiR5i|jgBW,Il^ ^^jn^gl| -- ' "I -- LIA11537 \ '' ' (' iSw iU-m TABLE 1 U S*n (^9 ft/ml tervw) URIS'ARY COPPER Saagla 1 3 3 4 $ 4 Ateaalc Ahaargtlaa a4 0.4 04 U a* u Cataef waSry 0.5 0.3 1.0 1.9 0.9 34 Recently tre were Heed to determine urinary copper la patients being treated for ilson'a disease. Thia disease it characterized by tbe palieot'a inability to metabolize ingested copper. The patients were bring treated with a chelating agent to cauae elimination of the organic copper complex through the urine. Urine aamplea were collected for a number of day*. Copper was determined directly against oxter ataodarda without any aample treatment. The aet of nine aamplea ai completed in aboot thirty minutes. ) On the first day, before treatment with the chelating agent, the urinary copper was very low as shown in Sample 1 of Table II. After ingestion of the drug, the copper rose to an abnormally high level, and then fell off gradually aa the body copper level began to return to normal. Similar aeries of data were obtained on two additional patients. ) : TAKE U Caggar la Urtaa* 7< Saogta Ca Ck/1) i 1 0.35 l 3 4.9 lf\ 3 U 4 4.9 * 4 i-. 1 15 t 4.9 7 49 045 * 0.17 * Sawglaa oatataay af D*. HaWi Ravin, Sinai Haagitol, Daw***, I2 'WfWfBMBHE spspff I. j' I '" ' "` v'7-' '( LI A1X5 36 : TV -\ : .'*r'H'K-J*H<`*-y '* -'*' '** - - i-i* ~w.... t --..w ._. V^y.-.v >-> .^.^a^w-ay.^YniTCTr f ZINC Tbe atomic ab*orption method it oat of tbe moat senaitive determination* of zinc. Many laboratories routinely uae atomic alaorptioa (o determine line in blood, urine and animtl titnuc (4). In n previou* Newtletter (6) e reported n very nimple eiperiment in which line was determined in urine without ntmple preparation ttept. TRACE ANALY SES IS UtilSE STTIIOUT SAMPLE PREPARATION i The atomic abaorption method can be utilized foe (Rany metala without preliminary temple treatment if t ipertmpbotottetef it used in conjunction with rvoine suppression circuit*, (a worh reported e*rli*r (3), etriout trtce metal* were determined ia blood and urine. Theaa determination* and the retulta of later wcci are tummariied ia Table III. TAIL! in Petactfea Ltwtr la Urtaa Metal Za Detectlea Unit 0.005 Or aoos Fa 005 Fh 0.15 Cd 001 O 001 Me 0.01 HI 0.05 ? C# 0.15 :' Tl 0.2 Me 0-2 r r Aw 0.1 A* 0.02 L Tbe arine wjje were determined witbout preparatory sample treatment lo all tbe aaalytea. Sample quantities of 5 ml of urine are (teoertlly recommeoded foe each delertninalloa, although 2 ml are unually sufficient. The entire analytical time for each element vanrt from 2 to 10 minutes per aample. depentfin* upon tbe number of aamplea to be determined Contldereblr prior eaperience indirate* that no Interference la etpeeted for the metals la Table 111 ia a urine malrii Some amall ( *> ii>i11ty of Interference etitta f< chromium and molybdenum, however, in both ce* recovery etperimenta ia urine Indicate no problem. = TRACE ANALYSES IN BLOOD Moat of tbe mrtala Itated ia Table III have aleo been determined in Mood down to concentration levela approximately <h>ide the detection limitaaliowa. Since blood I* too viaetjua ' :i 3 p u ju iiip w p y il'w a p ;u ? i wp y p w y p*uysrwr*'w:nsawf1.* *" 7 LIA11539 to be aspirated directly, the sarnie mutt first be treated If the metal it m>< bound to the protein or blood cell*, it ia particularly mote meat to dilute the blood 1:1 in 20% trichloroacetic acid. The sample is thee centrifuged and the i^nuiini run directly on the atomic absorption apparft* tu Standards are simple water solutions of the metals being determined. If the metal of interest ran be remold by precipitation, it will be necessary to < solve the blood ie acid, aa described ia the seexmd paj*er of thia Newalettef. Aa little 2 1 of blood will be required for either method of sample preparation. ~ < CHELATION* METHODS A nux^/er of worker* have remitted on the advantages of coupling simple chelation method* with atomic absorption spec trt>fhoioav*try U.5). Since the atomic absorption procedure ia specific for a particular metal, the chelation method need not be specific andthe main source of difficulty when chelation i* applied to coJonmetry ia therefore eliminated. Two important improvement % are effected when a aw*!*] i* chelated in the aqueous solution and extracted into nn organic advent. First, the sensitivity for each metal iaecbaaced about five times in most organ* ics that are immiscible in water, and second, the extrsrtinn procedure can be accomplished con* veflieatjy by adjusting the volume ratio between the aqueous and organic phases lo concentrate the metal in the organic phase. The sensitivity for unne samples can be increased more than twenty times with thia procedure when 23 el / urine per dcfemjioaiioa ia used. The sensitivity, for blood sae.pl ea can be i nr reaped about 10 times w ben 10 ml of blood is used. Copper and lead esn be chelated with aomoeiun pyrrolidine dithwx arl^amate (1. 8) and extracted into an organic solvent that ia immiscible is water. Many of the ketones are suitable. The etpmmeats! procedure be urine is quick and very simple. About 25 ml of acid* iTied sample. I ml of soluti^o of the chelating agent, and 5 ml of a suitable ketone are put , into a separatory funnel This empire is shuk'f; by hand for a frw minutr*, andthe fhavs are allowed to separate by standing for more than an hour. The aqjeous phase ia withdrawn from the funnel ind the organic phase i* collected in a lest tube. There is r*o need to collect the organic phase quantitative]* since the quantitative aspect is assured by the 5-lo*l ratio of sample to organic solvent. Other metal* that co-ettract produce no interference in the analysis. The standard* are similarly extracted iron simple aqueous solutions. Three replicate extra* turns f<r roj^er, in which the original aqueous solution coa* tamed 0 005 {pm roj^^r. are *k-.n ia Figure 1. fliere was a volume ratio of $ 1 between the organic and aqueous phases (>>{pef concentrations a* low as 0-5 fpb can be drteeted above bar kground A similar experiment with van*h i* concentrations of lead ia shown in Figure 2- Thia extends the detection limit to 0 01 {pm Fb m 14<od or urine, andthe analytical time ia about fifteen minutes j.e* ^termination The determination caa be performed with about 10 ml of b!<*>4* h LEAD IS' UfdNF. AND DL00D iffj n p iiiaq;i Aft nxperimrnf waa {reformed on a group <{ blond and urine samples from worhera : routinely ripoej to IrvJ The vcmr ape nurio *f * idi/iej lo al>wt |ll 2 9 using dilute 11(3 aid a |*lf mnrf as an indir a to# Twenty mlof r a< h unne sample were nd'Jrd to separatory funnels, (hie ml of 5% aqueous sedation of am#m*ium pyrmlidine dithitx srbamate and 10 ml of methyl iaolnjtyl ketone wefr adJed Jhe funnel* were shaken b/ hand for two minutes each Since aft emulsion formed at the iAlrffa< e of the layers, the sample* were centnfugrd to separate the phases, 0 ? 4 "qgEPJWjp., UyMMJ fly^.jrnyywyw11 LIAll540 fi| 2 . #11.0, 0 1 0.J ^ t+#d $ LIA1154T- Each aample contained about 6 ml of blood. An equal volume of 8% trichloroacetic acid waa added and the aample centrifuged. Si ml of the supernatant aolution wore brought to pH 2 8 by dropwiae addition of dilute NaOH One ml of the chelating aolution and 3 ml of ketone were added to the funnel*. The aamfdrs were shaken and centrifuged to separate the phases. The value* obtained fof the urine and blood a ample a ara given below. tampla 1 2 3 UHaaa <0.04 gp/ml 0.14 <0.04 Blaaia 0.34 14 There may be error a in (beae analyaea. For inatance. tba chelating agent may not recover all tbe oritanirally bound lead TKi a error would not be uncovered by Ailli*' expert merita (1) by recovery. Alao. it ia quite poaaible that in ike blood aamplea lead waa loat la the precipi tate. Thia potential error would hate been avoided if ike blood* bad been diaaolved ia nitric acid. e have not yet evaluated lbeae error*. ACKNOrLEDGEVENTS Ac wiak to thank the many laboratorie* with whom we have cooperated in ibeae ex periment*. Ac wiab l*o to acknowledge tbe contribution of D. C. Manning, who kaa performed some of the analttii *1 work Some of the extradion experiment* were conducted by flichard Thompson. who bit ia our laboratory for a few week*. REFERENCES I. J n Ailli*. Anal Chem 34.614 (1962}. 2 C. Feldman andH K. Dhumvad. Office Terh. Service*. TIO-76S3. Aacvbington. DC. 1963- 3. 4. Slavin, S. Sprague. F. Hieder*. V. Cordova. J. Forenaic Science*, (to appear), reprinted in tbi*Newsletter. 4 H E. Parker. Perkin Elmer Sewajettef Q, May 1963. 5 E Herman. Giniral fikemiral Congre.a*. Detroit, 1963. 6. Perkin-Elmer N'ewaletler So 11. March 1963. 7. II. Kabo andA. Slavin. Appd. Optica^. 931 (1963). 8 J.K. Allan, Sjectrochn* Ada 17, 459(1961). 9. Hanuiay. ASM. Ikocbrm. J, S3. 227 (1933). ?-g8aggUg t